(a) Field of the Invention
The present invention relates to a method for processing fractionated patterns of serum formed by the electrophoresis.
(b) Description of the Prior Art
FIG. 1 shows basic concentration distribution (densitogram) on fractionated patterns formed by electrically energizing with an electrophretic apparatus a carrier made of cellulose acetate film onto which man's serum is applied (a healthy man's serum generally shows such patterns). Such electrophoretic patterns usually consist of five fractions of A, B, C, D and E including five peaks located at a.sub.0, a.sub.1, a.sub.2, a.sub.3 and a.sub.4 corresponding to albumin, alpha 1 globulin, alpha 2 globulin, beta globulin and gamma globulin. Diagnosis or judgment whether or not the sample is normal or abnormal is done on the basis of such electrophoretic patterns and integrals or percentages of the individual fractions. However, electrophoretic densitograms actually obtained may include peaks produced by various causes in addition to the five shown in FIG. 1. The electrophoretic densitogram shown in FIG. 2, for example, has a peak located at a.sub.5 in addition to the normal five peaks. This peak is produced due to turbidity in serum which allows a substance insensible of electrophoresis to remain at the position of sample application. In another example, an additional peak may be produced at a different location in addition to the five basic peaks.
When colorimetry is done on a sample which shows peaks in addition to the five basic peaks, inconvenience is caused in automatic processing with a computer of data obtained by colorimetry. FIG. 3 shows an example of configuration of a densitometer and a photometric apparatus which are currently employed. In the block diagram shown in FIG. 3, the light emitted from a light source lamps 3 is passed through a lens 4, a filter 5 and a slit 6, used for irradiating a carrier 1 and detected with a photo detector element 7. The carrier 1 has fractionated patterns 2, 2', 2", . . . of sera formed thereon as shown in FIG. 4, and is placed between the light source and the detector for photometry of the individual fractionated patterns while scanning in the direction perpendicular to the shifting direction of the carrier. That is to say, the light emitted from the light source lamp and having passed through the sample applied onto the carrier is received by the photo detector element 7, whose output corresponding to sample concentration is amplified with a preamplifier 8, converted by a logarithmic converter 9 into logarithmic value and used for preparing analog patterns as shown in FIG. 1. Successively, output from the logarithmic converter 9 is inputted into an A/D converter 10 and converted at definite time intervals into a digital signal by operating a conversion command signal generator 11 with a photometry command 11a from a computer 12. Integral or percentage of each fraction is determined on the basis of the digital data obtained at this stage.
For the operations described above, it is sufficient to determine points of local minimum values as boundary points in such a case as shown in FIG. 1 for calculating integrals or percentages of the individual fractions. In case of an electrophoretic densitogram divided into more than five fractions as illustrated in FIG. 2, however, it is impossible to determine integrals, etc. of the five fractions since five or more boundary points exist. In a case where an electrophoretic densitogram has more than five fractions, it is therefore required for the analyst to check analog patterns and fractionated patterns, and perform recalculation through processing to attribute the additional peaks to any one of the albumin fraction, alpha 1 globulin fraction, alpha 2 globulin fraction, beta globulin fraction and gamma glubulin. In case of abnormal fractions due to disease, they may be reported with no attempt made for data processing.
In view of such circumstances, there have hitherto been developed methods, for example by Japanese patent application No. 64814/79 (U.S. patent application Ser. No. 151,889), for automatic data processing to attribute additional peaks to any one of the five basic fractions even when an electrophoretic densitogram has more than five fractions. The processing disclosed by the above-mentioned Japanese patent application will be described below. In the first place, a standard serum such as a commercially available control serum is analyzed by the electrophoresis to obtain electrophoretic densitogram having five fractions. On the electrophoretic densitograms, the peak locations and boundary points remain substantially unchanged so long as type of carriers and electrophoretic conditions are kept unchanged. Therefore, unknown samples to be analyzed for clinical inspections should show densitograms having development lengths nearly the same as that of the standard serum so long as the type of the carrier and electrophoretic conditions are kept the same.
Standard lengths (distances as measured from the origin to locations of the individual peaks and the individual boundary points on the x axis) are determined as described below. A densitogram as shown in FIG. 5 is obtained by photometry of the electrophoretic patterns. Data are sampled from the densitogram at constant time intervals and subjected to A/D conversion for storing concentrations at the sampling points. The sampling points are designated consecutively as 1, 2, 3, . . . n and plotted on the abscissa, and the concentrations at the sampling points are plotted along the ordinate. Based on the stored data, boundary points are to be detected. The boundary points have local minimum values on the densitogram. Let us therefore assume that an optional point on the densitogram has coordinates of x.sub.b and y.sub.b. Similarly, let us assume that neighboring points on the densitogram have coordinates of X.sub.b-1, y.sub.b-1 and x.sub.b+1, y.sub.b+1 respectively. Then, a boundary point can be located as point x.sub.b which satisfies the following relation: EQU y.sub.b &lt;y.sub.b-1, y.sub.b &lt;y.sub.b+1
Let us now consider the process to locate the points a.sub.0, a.sub.1, a.sub.2, a.sub.3 and a.sub.4 on the abscissa corresponding to peak tops. a.sub.0 should be located between the start point x.sub.0 and boundary point b.sub.1, a.sub.1 between the boundary points b.sub.1 and b.sub.2, a.sub.2 between the boundary points b.sub.2 and b.sub.3, a.sub.3 between the boundary points b.sub.3 and b.sub.4, and a.sub.2 between the boundary point b.sub.4 and end point x.sub.n. In the procedures similar to those used for determining the boundary points b.sub.1 through b.sub.4, a.sub.0 through a.sub.4 can be determined as x.sub.a corresponding to points on the densitogram which have y.sub.a values satisfying the following relation: EQU y.sub.a &gt;y.sub.a-1, y.sub.a &gt;y.sub.a+1
the values of b, b, . . . and a, a, . . . thus determined are proportional to the lengths as measured from the start point x.sub.0 to the points themselves in a relationship of 1:1. It is therefore possible to use a scale of constant time intervals in place of the lengths as measured from the start point.
The boundary points on the densitogram of the standard serum are determined as described above.
Successively, individual boundary points on a densitogram obtained on the basis of electrophoresis of an unknown sample are to be determined in the similar procedures. In case of a densitogram shown in FIG. 6 (B), for example, c.sub.1, c.sub.2, c.sub.3 . . . c.sub.8 are determined as boundary points. Using the a.sub.0, a.sub.1, a.sub.2 and a.sub.3 determined on the densitogram of the standard serum shown in FIG. 6 (A) as standard points, they are located along the abscissa of the densitogram of the unknown sample as shown in FIG. 6 (B). Number of boundary points is counted in each of the sections a.sub.0 -a.sub.1, a.sub.1 -a.sub.2, a.sub.2 -a.sub.3 and a.sub.3 -a.sub.4. When the number of boundary point is counted as 1, it is adopted as a normal boundary point. When two or more boundary points are counted, the one corresponding to the lowest concentration is adopted as a normal one and the others are erased. Speaking concretely with reference to FIG. 6 (B), only one boundary point exists in the section a.sub.0 -a.sub.1 and this is adopted as a normal boundary point. In the section a.sub.1 -a.sub.2, three boundary points c.sub.2, c.sub.3 and c.sub.4 exist and only c.sub.3 corresponding to the lowest concentration is adopted as a normal boundary point. In the section a.sub.2 -a.sub.3, only one boundary point c.sub.5 exists and this is adopted as a normal boundary point. In the section a.sub.3 -a.sub.4, c.sub.7 is selected as a normal boundary point out of the two boundary points c.sub.6 and c.sub.7. In addition, all the boundary points existing in the section from a.sub.4 to the end point are erased.
Boundary points produced by beta lipoprotein, beta 1.sub.c protein and foreign matters at the sample application positions always correspond to concentrations higher than those corresponding to the normal boundary points. Therefore, the above-described processing method is capable of correctly determining boundary points.
FIG. 7 shows a block diagram of the above-described conventional processing method. The conventional method will be described with reference to this block diagram. In the first place, a standard serum is measured with an electrophoretic apparatus 21. The measured values obtained are fed to a boundary point judging means 22 which determines peak locations (a.sub.0 through a.sub.4) and boundary points (b.sub.1 through b.sub.4) by the above-described method. These peak locations and boundary points are transferred to a standard position storing means 23 for storing reference data. Then, an unknown sample is measured with the electrophoretic apparatus 21, and on the basis of the measured values, the boundary point judging means 22 locates the points on the abscissa corresponding to the local maximum and minimum values on the densitogram. The values thus determined are sent to a five-fraction processing means 24 which compares the data with the values of a.sub.0, a.sub.1, . . . and b.sub.1, b.sub.2, . . . of the standard serum stored in advance in the standard position storing means 24 for determining correct boundary points. Once the correct boundary points have been thus located, it is possible to calculate integrals or percentage of the individual fractions.
The conventional processing method described above permits locating normal boundary points even when peaks and valleys exist on densitograms in addition to the normal peaks and normal boundary points. However, this method always requires standard sera which provide normal electrophoretic densitograms without fail. Since certain standard sera commercially available cannot always provide normal electrophoretic densitograms, it is rather tedious to select proper standard sera. In addition, even when proper standard sera are selected, they may be denatured depending on conditions at the storing site or contaminated by germs, thereby incapable of being developed into the correct five fractions. Therefore, storage of standard sera pose tedious problems.